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Glutaric Acidemia Type II via the ETFDH Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
2177 ETFDH$910.00 81479 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

Glutaric acidemia II patients are split between those with causative variants in the ETFA, ETFB and ETFDH genes, and it is difficult to estimate the fraction with variants in each gene. However, to date nearly all reported causative variants in the ETFDH gene are detectable via direct sequencing, and in studies with larger numbers of patients with ETFDH variants the majority had two variants identified. For example, Goodman et al. (2002) reported 36 ETFDH alleles in 20 patients, for an overall sensitivity of ~90%, while Olsen et al. (2007) reported 28 ETFDH alleles in 15 patients, for an overall sensitivity of ~93%. 

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 ETFDH$690.00 81479 Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

To date, only a single large deletion has been described in the ETFDH gene (Wen et al. 2010), although most studies of glutaric acidemia type II patients that included ETFDH analysis have not been reported to have included deletion and duplication testing.

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Clinical Features

Glutaric acidemia (GA) type II, also known as multiple acyl-CoA dehydrogenase deficiency (MADD), is an inherited disorder of fatty acid and amino acid oxidation. GA type II is caused by defects in one of three genes (ETFA, ETFB or ETFDH). Clinical and biochemical features are not typically useful in distinguishing which gene is affected in GA type II patients. Three sub-categories of GA type II are recognized, each of which is based on severity and presentation of clinical symptoms. (Frerman and Goodman 2014).                                                                                                                             

Patients in the first group present within the first 48 hours of life with severe hypoketotic hypoglycemia, hypotonia, metabolic acidosis, possibly hepatomegaly and a “sweaty feet” odor similar to that observed in isovaleric acidemia patients, and multiple congenital anomalies, such as dysplastic kidneys, facial dysmorphism, rocker bottom feet, abdominal wall defects and abnormal external genitalia. Such patients typically die within the first week of life (Olsen et al. 2003; Schiff et al. 2006; Frerman and Goodman 2014; Xi et al. 2014).  

Patients in the second group present similarly to those in the first group, but without congenital anomalies. If these patients survive beyond the first week of life, they typically succumb within the first few months, often due to cardiomyopathy (Olsen et al. 2003; Schiff et al. 2006; Frerman and Goodman 2014; Xi et al. 2014).

The patients in the third group have a more mild form of GA type II, with onset anywhere from infancy to adulthood. Clinical presentation also varies widely in this group, and may include vomiting, hypoglycemia, metabolic acidosis, hepatomegaly, progressive lipid storage proximal myopathy and exercise intolerance. Symptoms in the third group often present in an episodic manner, making biochemical analysis challenging as abnormalities may be detectable only during periods of metabolic crisis (Olsen et al. 2003; Schiff et al. 2006; Frerman and Goodman 2014; Xi et al. 2014).

In all GA type II patients, generalized aminoacidemia and aminoaciduria, hyperammonemia and metabolic acidosis may be observed. Increased levels of sarcosine in both the serum and urine are common in those with mild, later onset GA type II. Biochemically, GA type II can be distinguished from GA type I based on the presence of 2-hydroxyglutaric acid (3-hydroxyglutaric acid is present in GA type I patients) (Frerman and Goodman 2014).

To date, no effective treatments are available for patients with the severe, infantile onset forms of GA type II. Some patients with the mild, later onset form have been shown to respond well to riboflavin, glycine and L-carnitine supplementation, as well as to dietary restriction of fat and protein (Olsen et al. 2007; Wen et al. 2013; Frerman and Goodman 2014). The majority of patients reported with the late-onset, riboflavin responsive form of GA type II have had defects in the ETFDH gene (Olsen et al. 2007; Wen et al. 2010; Cornelius et al. 2012; Wen et al. 2013).

Genetics

Glutaric acidemia type II is an autosomal recessive disorder caused by pathogenic variants in the ETFA, ETFB or ETFDH genes. To date, approximately 130 causative variants have been reported in the ETFDH gene (Human Gene Mutation Database). The majority of reported pathogenic variants are missense, although nonsense, regulatory, and splicing variants, as well as small deletions, insertions, duplications and indels have been reported, as has one gross deletion (Human Gene Mutation Database; Wen et al. 2010). The variants are spread throughout the gene.

The ETFDH gene is located on chromosome 4 and contains 13 exons. ETFDH encodes the electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The ETF-QO protein is located in the inner mitochondrial membrane, where it accepts electrons transferred from the electron transfer flavoprotein (ETF) a/b heterodimer. These electrons are accepted from at least 12 other flavoprotein dehydrogenases, and are transferred from ETF-QO to the mitochondrial respiratory chain. Clinical symptoms observed in ETFDH deficient patients are due to a disruption of electron transfer to the respiratory chain (Cornelius et al. 2012; Frerman and Goodman 2014).

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the ETFDH gene plus ~20 bp of flanking non-coding DNA on each side. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.

Indications for Test

Individuals with a positive newborn screening result for glutaric acidemia type II are good candidates for this test, as are individuals that exhibit biochemical and clinical symptoms of GA type II. Family members of patients known to have ETFDH variants are also good candidates, and we will also sequence the ETFDH gene to determine carrier status.

Gene

Official Gene Symbol OMIM ID
ETFDH 231675
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Glutaric Aciduria, Type 2 AR 231680

Related Tests

Name
Glutaric Acidemia Type II Sequencing Panel
Glutaric Acidemia Type II via the ETFA Gene
Glutaric Acidemia Type II via the ETFB Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Cornelius N. et al. 2012. Human Molecular Genetics. 21: 3435-48. PubMed ID: 22611163
  • Frerman F.E. and Goodman S.I. 2014. Defects of Electron Transfer Flavoprotein and Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase: Glutaric Acidemia Type II. In: Valle D, Beaudet A.L., Vogelstein B, et al., editors. New York, NY: McGraw-Hill. OMMBID.
  • Goodman SI. et al. 2002. Molecular Genetics and Metabolism. 77: 86-90. PubMed ID: 12359134
  • Human Gene Mutation Database (HGMD).
  • Olsen R.K. et al. 2003. Human Mutation. 22: 12-23. PubMed ID: 12815589
  • Olsen R.K. et al. 2007. Brain. 130: 2045-54. PubMed ID: 17584774
  • Schiff M. et al. 2006. Molecular Genetics and Metabolism. 88: 153-8. PubMed ID: 16510302
  • Wen B. et al. 2010. Journal of Neurology, Neurosurgery, and Psychiatry. 81: 231-6. PubMed ID: 19758981
  • Wen B. et al. 2013. Molecular Genetics and Metabolism. 109: 154-60. PubMed ID: 23628458
  • Xi J. et al. 2014. Journal of Inherited Metabolic Disease. 37: 399-404. PubMed ID: 24357026
Order Kits
TEST METHODS

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org).  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Order Kits

Ordering Options


myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
REQUISITION FORM
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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